This procedure involves the use of hazardous chemicals (including embryotoxins). Review MSDS for information on exposure limit, health risks, first aid, handling, etc.
Wear appropriate Personal Protective Equipment for this procedure:
Lab coat
Nitrile gloves (double-layer; regularly check for holes)
Eye goggles
Mask (optional)
Plastic apron (optional)
Shoulder-length gloves (optional)
Place a piece of absorbent sheet on the work surface before starting the procedure.
Have all waste containers ready (see Clean-up).
5-ml cryo-tubes (e.g., ThermoScientific 5000-0050) labeled with date and contents (ask Masa for the labels)
purified water (e.g., Ricca Chemical 915025 or Fisher 9150-25)
sodium cacodylate trihydrate (solid; Ladd Research 20305)
glutaraldehyde (70% aqueous solution in 10-ml ampules stored at 4°C; Ladd Research 20108)
formaldehyde (20% aqueous solution in 10-ml ampules stored at 4°C; Ladd Research 20300)
calcium chloride dihydrate (CaCl2·2H2O)
magnesium sulfate heptahydrate (MgSO4·7H2O)
For microwave-assisted fixation:
1.5 tube per slice of 6% glutaraldehyde/2% formaldehyde (stored at -20°C in the freezer in NHB 3.360E)
tri-pour beaker
6-well plate or 35 mm plastic Petri dish (12-well plate can be used for mouse slices)
4 rings (used to make nets for interface chamber)
microwave oven with flatbed (no turn table)
Exhaust must be properly vented → see below for how to set it up.
We currently use a consumer model with 1500 W output, but also used one with 700 W output previously (Jensen & Harris, 1989)
an array of neon bulbs (e.g., EMS 97036-01)
[stock] or FW | [final] | Final volume = 350 ml | |
purified water to start with | - | - | ~225 ml |
Na cacodylate·3H2O | 214.03 g/mol | 0.1 M | 7.491 g |
CaCl2·2H2O | 0.4 M | 2 mM | 1.75 ml |
MgSO4·7H2O | 0.8 M | 4 mM | 1.75 ml |
Formaldehyde | 20% | 2% | 35.0 ml |
Glutaraldehyde | 70% | 6% | 30 ml |
Approximately 15-20 minutes before the end of your experiment, begin preparing for fixation.
Take 1.5 tube per slice of the fixative solution out of the freezer in NHB 3.360E.
Place tubes in a tri-pour beaker, place beaker in sink, and allow warm water to run over the tubes to thaw the fixative (should be warmed to 31°C).
Grab a 6-well plate on the shelf above the vibratome and 4 glass rings (ring only, no net) from the net-making supply bucket. Label the wells for the slices to be fixed.
Place each of the 4 rings in a separate well of the 6-well plate.
Place neon bulb array in microwave, and run microwave for ~30 seconds to warm up the magnetron. Make sure none of the bulbs light up (which indicates a hot spot). Take array out of microwave and leave door ajar. Set microwave to 20 seconds.
Time should be adjusted depending on the method of subsequent tissue processing.
Once fixative is warmed, add fixative with a transfer pipette to each of the 4 wells (one 5-ml tube per well).
After last data point is collected and the recording ends, remove the camera/caps, withdraw all of the electrodes out of the chambers and fully retract manipulators.
Bring 6-well plate with fixative over to the rig and starting with the first chamber, take off the cover, pick up net by edge using ephys tweezers, and then with help of fix tweezers (marked with red tape), flip the net over onto ring in 6 well plate so that slice is completely submerged, but not touching the bottom of the well.
Repeat step 9 with remaining 3 chambers. Note to be very CAREFUL of recording electrodes.
Place 6-well plate with all 4 slices in microwave, close door and press start.
After microwave is done, take 6-well plate out and add remaining tube of fixative to all 4 wells. Place lid back on plate and leave in the fume hood overnight.
Hazardous Liquid Waste: Pour all waste into the proper waste collection bottles available in the fume hood in NHB 3.360E.
Aldehyde-Cacodylate (fixative solution, cacodylate buffer)
Hazardous Solid Waste: Place all contaminated solid waste (e.g., gloves, tubes, processing dishes, etc.) into hazardous waste bags in the fume hood. All tubes must be uncapped.